MATRIEX Imaging: Multi-Area Two-photon Real-time In-vivo Explorer

Two-photon laser scanning microscopy, originally developed since 1990s, has been widely applied for biomedical research in recent decades, particularly popular among neuroscientists for studying neural functions in vivo. However, it is typically restricted to one imaging area that is orthogonal to the optical axis. Here, we demonstrate a novel multi-axis optical conjugation method that enables two-photon imaging at single-cell resolution simultaneously in multiple areas at different depths, each of which could have a view diameter of ~200 µm and could be largely freely targeted within a zone up to 12-mm diameter. For example, we show simultaneous imaging of neuronal activities in the primary visual cortex (V1), the primary motor cortex (M1) and the hippocampal CA1 region of awake mice. This method can be readily implemented on a single conventional two-photon microscope to enable multi-area exploration of neuronal activities in vivo.